[The study of mechanisms of accumulation of daunorubicin and rodamin-123 in cells of human venous blood using cytometry technique].

The article presents comparative data of cytometry estimation of accumulation of daunorubicin and rodamin-123 in cells of peripheralbloodofhealthypeople underincubation ofsubstances in vitro. It is demonstrated that maximal saturation of thrombocytes occurs during the first five minutes, of leukocytes during forty five minutes. The erythrocytes factually never accumulate these compounds. The maximal values of accumulation of substances in leukocytes are characterized by high inter-individual variation. The close correlation (Rs = 0.96-0.98) of parameters of accumulation of substances in lymphocytes and neutrophils testifies the presence ofsimilar mechanisms ofcontrol ofactivity transportation ofxenobiotics in nucleated cells of blood. However, the results of inhibitor analysis of input of Pgp-dependent mechanisms of accumulation of rodamin-123 by leukocytes differ the data received under application of daunorubicin that reflects differences of their intracellular binding sites. The expressed differences between parameters of accumulation ofdaunorubicin and rodamin-123 by leukocytes in various patients determine necessity of individual approach in monitoring of development of medicinal resistance.

Effects of hepatic ischemia-reperfusion injury on the P-glycoprotein activity at the liver canalicular membrane and blood-brain barrier determined by in vivo administration of rhodamine 123 in rats.

PURPOSE: To investigate the effects of normothermic hepatic ischemia-reperfusion (IR) injury on the activity of P-glycoprotein (P-gp) in the liver and at the blood-brain barrier (BBB) of rats using rhodamine 123 (RH-123) as an in vivo marker. METHODS: Rats were subjected to 90 min of partial ischemia or sham surgery, followed by 12 or 24 h of reperfusion. Following intravenous injection, the concentrations of RH-123 in blood, bile, brain, and liver were used for pharmacokinetic calculations. The protein levels of P-gp and some other transporters in the liver and brain were also determined by Western blot analysis. RESULTS: P-gp protein levels at the liver canalicular membrane were increased by twofold after 24 h of reperfusion. However, the biliary excretion of RH-123 was reduced in these rats by 26%, presumably due to IR-induced reductions in the liver uptake of the marker and hepatic ATP concentrations. At the BBB, a 24% overexpression of P-gp in the 24-h IR animals was associated with a 30% decrease in the apparent brain uptake clearance of RH-123. The pharmacokinetics or brain distribution of RH-123 was not affected by the 12-h IR injury. CONCLUSIONS: Hepatic IR injury may alter the peripheral pharmacokinetics and brain distribution of drugs that are transported by P-gp and possibly other transporters.

Contribution of radixin to P-glycoprotein expression and transport activity in mouse small intestine in vivo.

The ERM proteins, ezrin, radixin, and moesin, are membrane-cytoskeleton cross-linkers with multiple physiological functions. We previously showed that radixin is involved in posttranslational regulation of P-glycoprotein (P-gp) in human hepatoblastoma HepG2 cells. Here, we investigated the physiological role of radixin in regulating P-gp expression and activity in the small intestine by comparing wild-type- and radixin knockout (Rdx) mice. In intestinal tissue homogenates, P-gp protein levels increased markedly from the upper part to the lower part of the small intestine in both wild-type- and Rdx(-/-) mice. In the membrane fractions, a similar pattern was seen in wild-type mice. However, the membrane expression of P-gp protein remained at the same level from the upper to the lower part of the small intestine in Rdx(-/-) mice. When rhodamine123 (Rho123), a substrate of P-gp, was orally administered to Rdx(-/-) and wild-type mice, the absorption phase of Rho123 was greater in Rdx(-/-) than in wild-type mice, whereas the elimination phase in Rdx(-/-) mice was not different from that of wild-type mice. Our results indicate that radixin plays an important role in regulating P-gp localization and P-gp functional activity at the intestinal membrane.

Changes in absorption and excretion of rhodamine 123 by sodium nitroprusside.

Nitric oxide (NO) donors increase the permeability of water-soluble compounds with neither loss of cell viability nor lactate dehydrogenase release, but the involved mechanism is not fully understood. In this study, we focused on permeation via the transcellular route and P-glycoprotein, which is a typical ABC transporter. We examined the effect of sodium nitroprusside (SNP), which is an NO donor, on the membrane permeation of rhodamine 123 (Rho123), a representative P-gp substrate, and the change in expression level of ileal P-gp. We used an in situ closed loop method in rat ileum to study changes in the permeation of Rho123. The effects of SNP (1 and 10mg/kg) on the mdr-1a mRNA and P-gp protein expression levels were examined by real-time RT-PCR and Western blotting, respectively. The absorption and excretion of Rho123 were significantly increased in an SNP dose-dependent manner when compared with those with no addition, but no changes in protein expression level of P-gp in ileal BBM were observed by SNP administration. The relative activity of P-gp was not changed by SNP administration. On the other hand, the expression level of mdr-1a mRNA was induced by SNP administration. We indicated that SNP could increase the mucosal permeation of Rho123 via the transcellular route without an influence on P-gp, and we showed that this effect is temporary. SNP has no influence on P-gp function and protein expression level in the short term, but they may change in the long term.

The role of inter-segmental differences in P-glycoprotein expression and activity along the rat small intestine in causing the double-peak phenomenon of substrate plasma concentration.

  Conflicting results have been reported on segmental differences in expression of P-glycoprotein (P-gp) along the small intestine of animals and humans. In this study, we investigated P-gp mRNA and protein levels within each of nine segments of rat small intestine. In addition, P-gp activity in each segment was evaluated in terms of permeability of rhodamine123 (Rho123), a typical P-gp substrate, using the serial intestinal non-everted sac method. The P-gp mRNA levels tended to increase from the duodenum to the ileum, with peaks in the upper and lower ileum, while P-gp protein level reached its maximum in the middle ileum. The activity of P-gp was also the highest in the middle ileum, and was highly correlated with P-gp protein level. The double-peaked plasma concentration profile that was observed following oral administration of Rho123 to rats could be well reproduced by an intestinal compartmental kinetic model incorporating inter-segmental differences of absorption and excretion rate constants. Our results suggest that the heterogeneous distribution of P-gp along the small intestine plays a key role in causing the double-peak of plasma concentration of P-gp substrates following oral administration to rats.

P-glycoprotein and multidrug resistance-associated protein 2 are oppositely altered in brain of rats with thioacetamide-induced acute liver failure.

BACKGROUND: P-glycoprotein (P-GP) and multidrug resistance-associated protein 2 (MRP2) are involved in transport of many drugs across blood-brain barrier (BBB). The function and expression of P-GP and MRP2 may be modulated by different pathologies. Acute liver failure (ALF) was reported to impair BBB function, resulting in the increased BBB permeability. AIMS: We investigated whether ALF altered function and expression of P-GP and MRP2 in brain of thioacetamide-induced ALF rats. METHODS: ALF was induced by intraperitoneal injection of thioacetamide (300 mg/kg) for 2 days with a 24-h interval. The rats were used for experiments at 6, 12 and 24 h after the second administration. P-GP and MRP2 function in brain were determined using the brain-to-plasma ratios of corresponding substrates (rhodamine 123 and vincristine for P-GP; sulfobromophthalein and dinitrophenyl-S-glutathione for MRP2). Evans blue was used for examining the BBB integrity. Western blot was accomplished to determine P-GP and MRP2 protein expression. RESULTS: The brain-to-plasma ratios of rhodamine 123 and vincristine were significantly increased in ALF-6 h rats and almost returned to normal levels in ALF-24 h rats, whereas those of sulfobromophthalein and dinitrophenyl-S-glutathione were decreased in all ALF rats. Western blot results showed that ALF decreased brain P-GP levels at 6 and 12 h, whereas increased MRP2 levels at 6, 12 and 24 h. No significant difference of Evans blue concentrations in brain was found among the four groups. CONCLUSIONS: Function and expression of P-GP and MRP2 in brain of thioacetamide-induced ALF rats were oppositely altered.

Determination of Rhodamine 123 in rat plasma utilizing liquid chromatography-tandem mass spectrometry.

Rhodamine 123 (R123), as a typical of P-gp substrate, was widely used to quantify P-glycoprotein (P-gp) functional efflux activity in vivo. A new, rapid and sensitive method was developed for quantifying R123 in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). R123 and Rhodamine 6G (R6G, the internal standard, IS) were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4:1) as the solvent and chromatographic separation was performed using a Zorbax Eclipse Plus C18 column. The mobile phase was composed of A: ammonium formate-formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid and B: methanol (A:B, 5:95, v/v). To quantify R123 and IS respectively, multiple reaction monitoring (MRM) transition of m/z 345.2→285.2 and m/z 443.3→415.2 was performed. The analysis time was 4 min in positive mode; the calibration curve was linear in the concentration range of 1-200 ng/ml. The lowest limit of quantification (LLOQ) reached 1 ng/ml. The intra and inter-day precision were less than 9.2% for the low quality control (QC) level, and 3.4% for other QC levels, respectively, while the intra and inter-day relative errors ranged between -7.4% and 9.1% for three QC concentration levels. The LC-MS/MS method proved to be simple, accurate, reliable and with a shorter running time and has been successfully applied to evaluate the functional activity of P-glycoprotein in an absorption experiment in the rat.

Effects of chronic renal failure on kidney drug transporters and cytochrome P450 in rats.

Chronic renal failure (CRF) leads to decreased drug renal clearance due to a reduction in the glomerular filtration rate. However, little is known about how renal failure affects renal metabolism and elimination of drugs. Because both depend on the activity of uptake and efflux by renal transporters as well as enzymes in tubular cells, the purpose of this study was to investigate the effects of CRF on the expression and activity of select renal drug transporters and cytochrome P450. Two groups of rats were studied: control and CRF (induced by 5/6 nephrectomy). Compared with control rats, we observed reductions in the expression of both protein and mRNA of Cyp1a, sodium-dependent phosphate transport protein 1, organic anion transporter (Oat)1, 2, and 3, OatK1/K2, organic anion-transporting polypeptide (Oatp)1 and 4c1, P-glycoprotein, and urate transporter 1, whereas an induction in the protein and mRNA expression of Mrp2, 3, and 4 and Oatp2 and 3 was observed. Cyp3a expression remained unchanged. Similar results were obtained by incubating a human proximal tubule cell line (human kidney-2) with sera from CRF rats, suggesting the presence of uremic modulators. Finally, the renal elimination of [(3)H]digoxin and [(14)C]benzylpenicillin was decreased in CRF rats, compared with controls, as shown by a 4- and 9-fold accumulation, respectively, of these drugs in kidneys of rats in CRF. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs and reduced renal clearance that could, at least partially, explain the tubular toxicity of many drugs.

Effect of lipopolysaccharide on P-glycoprotein-mediated intestinal and biliary excretion of rhodamine123 in rats.

The effects of lipopolysaccharide (LPS) on the ileal and biliary excretion of rhodamine123 were investigated in rats at different times after intraperitoneal (i.p.) injection (1 mg/kg and 5 mg/kg of body weight). P-gp protein decreased 8h after injection of LPS and returned to the control level 24h after i.p. injection of LPS in the ileum. There was a marked decrease in the expression level of mdr1a mRNA in the ileum and liver 8h after i.p. injection of LPS when compared with the control condition. Also, the ileal and biliary clearance of rhodamine123 significantly decreased 8h after i.p. injection of LPS, but returned to the control levels 24h after i.p. injection of LPS. These results suggest that LPS-induced decreases in P-gp-mediated ileal and biliary excretion of rhodamine123 were probably due to impaired P-gp-mediated transport ability. The levels of iNOS and IL-1beta mRNA in the ileum and liver increased 2 and 8h after i.p. injection of LPS, respectively, and returned to the control levels 24h after injection of LPS. These findings suggest that LPS markedly decreases P-gp-mediated ileal and biliary excretion of rhodamine123, probably by partly decreasing the expression of P-gp protein levels, likely due to increased lipid peroxidation levels through iNOS mRNA and inflammatory mediators such as IL-1beta.

Effects of lipopolysaccharide on P-glycoprotein expression and activity in the liver and kidneys.

There have been many reports that P-glycoprotein expression and activity are altered during sepsis, but few of them have examined such changes over 72 h. In this study, we examined the effect of lipopolysaccharide (LPS, 5mg/kg, ip) on P-glycoprotein expression (Western blotting) and activity (rhodamine-123 (Rho123) pharmacokinetics) in liver and kidneys for 7 days. On day 1 after LPS administration, hepatic P-glycoprotein expression and activity significantly decreased. On day 3, hepatic P-glycoprotein expression significantly increased compared with the control group, while activity had returned to the control level. On day 7, hepatic P-glycoprotein expression returned to the control level. There were no significant changes in P-glycoprotein expression or activity in the kidneys after LPS administration. The amount of Rho123 excretion in urine remained unchanged with (4.2%) or without (4.0%) LPS administration, but the amount of Rho123 excretion in bile decreased from 2.0 to 0.7% with LPS administration. Our findings suggested that hepatic P-glycoprotein expression and activity decreased on day 1 but recovered within 3 days, but there were no significant differences in the kidneys after LPS administration. These results suggested that the change in P-glycoprotein activity might be due to change in P-glycoprotein expression in the liver rather than the kidneys.

Combined effects of epileptic seizure and phenobarbital induced overexpression of P-glycoprotein in brain of chemically kindled rats.

BACKGROUND AND PURPOSE: The multidrug resistance of epilepsy may result from the overexpression of P-glycoprotein, but the mechanisms are unclear. We investigated whether the overexpression of P-glycoprotein in the brains of subjects with pharmacoresistant epilepsy resulted from both drug effects and seizure activity. EXPERIMENTAL APPROACH: Kindled rats were developed by injecting a subconvulsive dose of pentylenetetrazole (33 mg.kg(-1).day(-1), i.p.) for 28 days. Groups were then treated with an oral dose of phenobarbital (45 mg x kg(-1) x day(-1)) for 40 days. In accord with behavioural observations, P-glycoprotein activity in brain was assessed using brain-to-plasma concentration ratios of rhodamine 123. P-glycoprotein levels in the brain regions were further evaluated using RT-PCR and Western blot analysis. The distribution of phenobarbital in the brain was assessed by measuring phenobarbital concentrations 1 h following its oral administration. KEY RESULTS: The kindling significantly increased P-glycoprotein activity and expression. Good associations were found among P-glycoprotein activity, expression and phenobarbital concentration in the hippocampus. Short-term treatment with phenobarbital showed good anti-epileptic effect; the maximum effect occurred on day 14 when overexpression of P-glycoprotein was reversed. Continuous treatment with phenobarbital had a gradually reduced anti-epileptic effect and on day 40, phenobarbital exhibited no anti-epileptic effect; this was accompanied by both a re-enhancement of P-glycoprotein expression and decreased phenobarbital concentration in the hippocampus. P-glycoprotein function and expression were also increased in age-matched normal rats treated with phenobarbital. CONCLUSIONS AND IMPLICATIONS: The overexpression of P-glycoprotein in the brain of subjects with pharmacoresistant epilepsy is due to a combination of drug effects and epileptic seizures.

Effect of cyclosporine on drug transport and pharmacokinetics of nifedipine.

Nifedipine (NFP) is an anti-hypersensitive drug and a well-known substrate of cytochrome P450 3A4 (CYP3A4), while cyclosporine (CSP) is a potent p-glycoprotein (P-gp) inhibitor. P-gp is a drug transporter, which determines the absorption and bioavailability of many drugs that are substrates for P-gp. Drugs that induce or inhibit P-gp may have a profound effect on the absorption and pharmacokinetics (PK) of drugs transported by P-gp within the body, possibly compromising their bioavailability. But the role of P-gp in the NFP efflux and its impact on PK profile is not known. Hence in our present study we attempted to investigate the effect of CSP on oral absorption and PK of NFP. Rhodamine 123 (Rho 123), a known P-gp substrate was used as a positive control. Male Wistar rats (350-400 g) were used for the study. Rats were divided into 4 groups (n=6 each); one group was treated with vehicle (cremophor) followed by NFP (0.2 mg/kg; i.v. bolus) and the other group with CSP (10 mg/kg; i.v.) followed by NFP. Group 3 and 4 were treated with vehicle (cremophor) followed by Rho 123 (0.2 mg/kg, i.v.) and CSP (10 mg/kg; i.v.) followed by Rho 123 (0.2 mg/kg, i.v.) respectively. The blood samples were collected at 0, 5, 10, 15, 30, 60, 90, 120, 180 and 240 min after NFP administration. NFP concentrations in plasma were analyzed by LC-MS-MS and Rho 123 was analyzed by fluorimetric detector. NFP efflux was significantly decreased in CSP treated rats (49.1% decrease, P<0.05), while NFP concentration in plasma were not changed. However the decrease in NFP efflux did not show any significant changes in NFP PK parameters (T(max); 2.0 vs. 2.5 min, C(max); 0.084 vs. 0.076 microg/ml, T(1/2); 84.0 vs. 91.4 min, AUC(0-t); 4.183 vs. 3.467 microg h/ml, AUC(infinity); 5.915 vs. 4.769 microg h/ml, AUMC(0-t); 224.073 vs. 173.063 microg h/ml, AUMC(infinity); 776.871 vs. 575.038 microg h/ml, MRT(0-t); 53.608 vs. 49.538 microg h/ml, MRT(infinity); 118.194 vs. 115.246 microg h/ml, CL(tot); 0.0375 vs. 0.0433 l/h, Vd(ss); 3.999 vs. 4.641 l in NFP alone vs. CSP+NFP groups respectively). Thus the results indicate that NFP would belong to a group of P-gp substrate. The decrease in efflux of NFP by CSP, through inhibition of P-gp, into the intestinal lumen did not show any impact on PK. This could be due to the activity of other transporters and/or CYP3A4 may have more limiting role than P-gp on NFP metabolism and disposition that is why inhibiting P-gp did not lead to increase the bioavailability and PK alterations.

In vivo comparison of various polymeric and low molecular mass inhibitors of intestinal P-glycoprotein.

Several polymers have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a direct in vivo comparison of delivery systems based on Pluronic P85, Myrj 52 and chitosan-4-thiobutylamidine (Ch-TBA) in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. Furthermore, the postulated low molecular mass P-gp inhibitors 6-mercaptopurine and reduced glutathione (GSH) were evaluated in vitro and in vivo. In vitro, the permeation enhancing effect of 6-mercaptopurine, GSH, Pluronic P85, Myrj 52, and the combination of Ch-TBA with GSH was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type diffusion chambers. In comparison to buffer only, Rho-123 transport in presence of 100 microm 6-mercaptopurine, 0.5% (w/v) GSH, 0.5% (w/v) Pluronic P85, 0.5% (w/v) Myrj 52 and the combination of 0.5% (w/v) Ch-TBA/ 0.5% (w/v) GSH, was 2.1, 1.6, 1.9, 1.8, 3.0-fold improved, respectively. In vivo in rat, enteric-coated tablets based on Pluronic P85, Myrj 52 or Ch-TBA/GSH increased the area under the plasma concentration time curve (AUC(0-12)) of Rho-123 1.6-fold, 2.4-fold, 4.3-fold, respectively, in comparison to control only. Contrariwise, the low molecular mass excipients 6-mercaptopurine and GSH showed no significant effect in vivo at all. This in vivo study showed that polymeric P-gp inhibitors and especially the delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.